Abstract
Mesenchymal stem cells (MSC) are critical components of the bone marrow (BM) niche, essential for maintaining the hematopoietic stem cell (HSC) pool. BM resident HSCs undergo diurnal oscillations in differentiation and release to circulation, contributing to daily cycles of blood and immune cell production, while replenishing the HSC reservoir at night. In parallel, BM-MSCs give rise to new bone forming osteoblasts and sustain their own pool via circadian bone turnover processes that remain incompletely understood.
Here, we investigated whether BM-MSCs undergo circadian changes in metabolism and function. Functional assays for colony forming units-fibroblasts (CFU-F) and osteoblast progenitors (CFU-Ob) revealed enhanced MSC proliferation and osteogenic differentiation potential at night, coinciding with peak levels of melatonin in the BM. Exogenous melatonin administered during the day also elevated their CFU-Ob potential, suggesting melatonin enhances osteogenic potential in a time-of-day dependent manner. Additionally, MSCs exhibited elevated cell and nuclear size at night, in contrast to primitive HSCs (pHSC), which shrink during this period. Both stem cell types exhibit improved functional potency at night when the smaller HSCs have improved BM competitive repopulation potential, while the larger MSCs have elevated proliferation and differentiation potential.
CD51+ MSCs, which have higher osteogenic and adipogenic potential, were upregulated in a circadian manner by Wnt signaling. Daytime Wnt activation increased CD51 expression, while nighttime inhibition suppressed it, indicating that Wnt is a key circadian driver of MSC identify and function. Administration of the Wnt agonist, BIO or expression of the Wnt-activating mutant, APCmin/+ transgene in mice increased MSC size at night, supporting the role of Wnt signaling in regulating MSC morphology and osteogenic potential under circadian control.
Mechanistically, the bioactive lipid, sphingosine 1-phosphate (S1P), an important regulator of HSC migration, was also implicated in MSC rhythmicity. Reduced S1P signaling in SPHK1-deficient mice disrupted the diurnal MSC size change. These alterations support the notion that S1P signaling is another essential metabolic regulator of BM-MSC morphology and developmental potential, instrumental in driving MSC bone-forming ability.
Despite clear functional and morphological circadian shifts and considering diurnal functional changes are closely regulated by cell metabolic state, BM-MSCs showed no time-of-day differences in glucose uptake nor mitochondrial membrane potential under homeostatic conditions. However, they exhibited lower mitochondrial mass at night, unaccompanied by changes in mitochondrial number. These observations suggest circadian alterations in mitochondrial dynamic state, paralleling those documented in pHSCs. Given the established links between sphingolipids and mitochondrial morphology, we explored metabolic changes additionally in SPHK1-KO mice. SPHK1 deficiency selectively increased morning glucose uptake in BM-MSCs as opposed to wild type mice. These data implicate that MSC metabolism, especially during daytime are potentially linked to sphingolipid metabolism.
Altogether, these findings identify a circadian interplay between melatonin, Wnt and S1P signaling pathways in regulating BM MSC morphology, mitochondrial dynamics, and osteogenic potential. These findings suggest a novel, coordinated circadian crosstalk between MSC and pHSCs essential for BM homeostasis and stem cell maintenance, with potential translational opportunities to enhance bone regeneration, BM stem cell function and counteract age-related decline in skeletal and hematopoietic health.
